Difference between revisions of "Part:BBa K4361300"
 
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<partinfo>BBa_K4361300 short</partinfo>
 
<partinfo>BBa_K4361300 short</partinfo>
  
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A mutant of the BlcR protein, created through site-directed mutagenesis with primers R1 ([[Part:BBa_K4361200]]) and F1.1 D37R ([[Part:BBa_K4361201]]). For this mutant, the aspartic acid in position 37 has been changed to arginine by mutating the GAC codon to CGC.
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A mutant of the BlcR protein ([[Part:BBa_K4361100]]), created through site-directed mutagenesis with primers R1 ([[Part:BBa_K4361200]]) and F1.1 D37R ([[Part:BBa_K4361201]]). For this mutant, the aspartic acid in position 37 has been changed to arginine by mutating the GAC codon to CGC.
  
 
This mutant also contains the following nucleotide mutations outside of the targeted site:
 
This mutant also contains the following nucleotide mutations outside of the targeted site:
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<h3>Usage and Biology</h3>
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<h3>Experimental results</h3>
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The set of BlcR mutants (this part through </html>[[Part:BBa_K4361319]]<html>) were expressed in a PURE system in the presence of GreenLys (fluorescent Cy2-labeled lysine). Samples were then run on an SDS PAGE gel (<b>Figure 1.</b>) to determine whether or not the protein was produced. For this mutant (<u>lane 1</u>), BlcR mutant D37R could not be succesfully produced in the PURE system.
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<a href="https://static.igem.wiki/teams/4361/wiki/results/greenlys.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/greenlys.png" style="width:600px;margin-left:125px"></a>
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<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-1.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-1.png" style="width:600px;margin-left:125px"></a>
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<figcaption> <b>Figure 1.</b> SDS PAGE gel of PURE reactions with GreenLys (fluorescent Cy2-labeled lysine) and a variant of </html>[[Part:BBa_K4361106]]<html> containing a BlcR mutant (this part through </html>[[Part:BBa_K4361319]]<html>). Arrows indicate bands corresponding to BlcR dimers and monomers, respectively. C = negative control, WT = wildtype BlcR. This part corresponds to lane <u>1</u>.</figcaption>
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<figcaption> <b>Figure 1.</b> SDS PAGE showing the translation products of 20 different plasmids coding for BlcR mutants. PURE reaction solution was supplemented with GreenLys and the synthesized proteins were imaged with Typhoon using a 488-nm laser. C: negative control with no plasmid. WT: positive control with expressed wildtype BlcR. 1: D37R mutant. </figcaption>
 
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Latest revision as of 18:14, 13 October 2022


BlcR D37R

A mutant of the BlcR protein (Part:BBa_K4361100), created through site-directed mutagenesis with primers R1 (Part:BBa_K4361200) and F1.1 D37R (Part:BBa_K4361201). For this mutant, the aspartic acid in position 37 has been changed to arginine by mutating the GAC codon to CGC.

This mutant also contains the following nucleotide mutations outside of the targeted site:

  • G 763 > A, resulting in substitution E255K

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 694
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 78
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 589

Experimental results

The set of BlcR mutants (this part through Part:BBa_K4361319) were expressed in a PURE system in the presence of GreenLys (fluorescent Cy2-labeled lysine). Samples were then run on an SDS PAGE gel (Figure 1.) to determine whether or not the protein was produced. For this mutant (lane 1), BlcR mutant D37R could not be succesfully produced in the PURE system.
Figure 1. SDS PAGE showing the translation products of 20 different plasmids coding for BlcR mutants. PURE reaction solution was supplemented with GreenLys and the synthesized proteins were imaged with Typhoon using a 488-nm laser. C: negative control with no plasmid. WT: positive control with expressed wildtype BlcR. 1: D37R mutant.